The broad, long term objective of this proposal is to determine the mechanism through which the yeast Ras guanine nucleotide exchange factor, Cdc25p, transduces the signal from glucose stimulation into the yeast Ras pathway. The specific aims are: 1. to delineate the functional and activation of Cdc25p; and 2. to analyze the role orf vesicular transport in regulation of the yeast Ras-mediated glucose response. The Ras signaling pathway couples environmental conditions with the regulation of cellular proliferation. Ras pathway components are structurally and functionally conserved from yeast to man. Deregulation of this pathway results in uncontrolled proliferation, which in mammals, can lead to cancer. Because of the evolutionary conservation, it is advantageous to study this pathway in the genetically tractable yeast. The function and activation of Cdc25p will be studied by mapping the regulatory domains of Cdc25p using mutagenesis. This domain will be used to identify potential components of the Ras pathway functioning upstream of Cdc25p through affinity chromatography and the yeast two-hybrid system. Potential upstream components will also be identified using a genetic screen. Candidate proteins identified using these techniques will be further characterized using genetic and biochemical techniques. The role of vesicular transport in transducing the glucose signal into the Ras pathway will be analyzed by tracking the subcellular localization of Cdc25p using yeast strains containing mutations in the secretory pathway and by mapping the subcellular localization domains present in Cdc25p by mutagenesis.